It is well recognized that physiological blood coagulation requires the presence of membranes composed of negatively charged phospholipids. Zymogen activations occur rapidly when the enzyme, usually a vitamin K dependent protein, binds to a cofactor, usually a non-vitamin K dependent protein, to activate a substrate, usually a vitamin K dependent protein, reviewed in (Mann, K. G., Jenny R. J., and Krishnaswamy, S. 1988. “Cofactor proteins in the assembly and expression of blood clotting enzyme complexes,” Ann. Rev. Biochem. 57:915-956; and Furie, B. and Furie, B. C. 1988. “The molecular basis of blood coagulation,” Cell. 53:505-518). These reactions include those that are both procoagulant and those that are anticoagulant.
In addition to a net negative charge, the nature of the phospholipid head group appears to contribute to catalytic and binding efficiency. Phosphatidylserine (PS) is generally considered to be the most important phospholipid (Pei, G., Powers, D. D., and Lentz, B. R. 1993. “Specific contribution of different phospholipid surfaces to the activation of prothrombin by the fully assembled prothrombinase,” J. Biol. Chem. 268:3226-3233; and Mann, K. G., Jenny R. J., and Krishnaswamy, S. 1988. Ann. Rev. Biochem. 57:915-956). It has been found that the presence of phosphatidylethanolamine (PE) or cardiolipin potently enhanced the rate of inactivation of factor Va by the activated protein C (APC) complex (Smirnov, M. D. and Esmon, C. T. 1994. “Phosphatidylethanolamine incorporation into vesicles selectively enhances factor Va inactivation by activated protein,” C. J. Biol. Chem. 269:816-819). APC is a critical natural anticoagulant required for preventing lethal thrombosis. The inactivation of factors Va and VIIIa by the APC complex is crucial in the regulation of coagulation, as evidenced by the severe clinical problems which are observed if this reaction is compromised (Esmon, C. T. and Schwarz, H. P. 1995. “An update on clinical and basic aspects of the protein C anticoagulant pathway,” Trends Cardiovasc. Med. 5:141-148). The presence of unsaturated fatty acids in the phospholipid vesicles also has been found to enhance the inactivation of factor Va (Smirnov, M. D., Ford, D. A., Esmon, C. T., and Esmon, N. L. 1999. “The effect of membrane composition on the hemostatic balance,” Biochemistry 38:3591-3598). A role for PE in factor VIII binding (Gilbert, G. E. and Arena, A. A. 1995. “Phosphatidylethanolamine induces high affinity binding sites for factor VIII on membranes containing phosphatidyl-L-serine,” J. Biol. Chem. 270:18500-18505), tissue factor-factor VIIa activation of factor X (Neuenschwander, P. F., Bianco-Fisher, E., Rezaie, A. R., and Morrissey, J. H. 1995. “Phosphatidylethanolamine augments factor VIIa-tissue factor activity: enhancement of sensitivity to phosphatidylserine,” Biochemistry 34:13988-13993) and prothrombin activation (Smeets, E. F., Comfurius, P., Bevers, E. M., and Zwaal, R. F. A. 1996. “Contribution of different phospholipid classes to the prothrombin converting capacity of sonicated lipid vesicles,” Thromb. Res. 81:419-426; and Billy, D., Willems, G. M., Hemker, H. C., and Lindhout, T. 1995. “Prothrombin contributes to the assembly of the factor Va-factor Xa complex at phosphatidylserine-containing phospholipid membranes,” J. Biol. Chem. 270:26883-26889) has also been reported. In those latter studies, the PE effects were different both qualitatively and quantitatively from those on the APC complex (Sec (Smirnov, M. D., Ford, D. A., Esmon, C. T., and Esmon, N. L. 1999. Biochemistry 38:3591-3598) for discussion).
One group of patients who are at increased risk for thrombotic diseases are those who have lupus anticoagulants, which are antibodies which bind to anionic phospholipids used in clotting assays based on the PTT (partial thromboplastin time) or APTT (activated partial thromboplastin time) techniques. See The Merck Manual (16th Ed. 1992) at 1225; J. E. Ansell, Handbook of Hemostasis and Thrombosis (Little, Brown & Co., Boston) at 19 (1986). Typical PTT test results for patients having the lupus anticoagulant are a prolonged clotting time that fails to correct with a 1:1 mixture of the patient's and normal plasma, a normal or minimally prolonged PT (prothrombin time), and a nonspecific depression of those clotting factors measured by a PTT technique (Factors XII, XI, X and VIII). The lupus anticoagulant antibodies may also react with cardiolipin which can interfere with assays utilizing cardiolipin as a reagent. See The Merck Manual, supra. Anti-cardiolipin or anti-phosphatidylethanolamine antibodies can cross react with each other, but not interact with sufficient affinity to procoagulant phospholipids to be anticoagulants. Because of the specificity of the APC complex for phospholipids, such antibodies would selectively inhibit APC anticoagulant activity without influencing the coagulation tests performed in the absence of APC which are used to diagnose the presence of a “lupus anticoagulant.”
Despite interference of the lupus anticoagulant antibodies with procoagulant phospholipid in clotting tests in vitro, persons with the antibodies have been reported to have an increased risk for thrombosis, either venous or arterial. Further, repeated spontaneous abortions in the first trimester of pregnancy have also been reported. Id. Patients have been treated with long term anticoagulant therapy to reduce the possibility of thrombosis, but no adequate technique has been developed for monitoring the effectiveness of such therapy. It should also be noted that other patients, who do not necessarily test positively for the lupus anticoagulant, may also be at risk for thrombotic disease due to the presence of antibodies not detected by current clotting tests. Further, not all persons who have the lupus anticoagulant or other risk factors have an identical propensity for thrombosis.
In order to attempt to identify patients at risk for thrombosis, standard clotting tests have been performed on patient plasma. Because anticoagulant therapy carries significant risk in some patients, it is highly desirable to determine whether patients are likely to benefit from such therapy. Additional testing has been suggested as described above where PTT and/or PT test results do not appear to be normal, such as repeating the test with added normal plasma or further addition of excess phospholipid. Previously, a technique was developed to differentiate among lupus patients and among others which patients have the highest propensity to have a thrombotic incident. This method was disclosed and claimed in U.S. Pat. No. 5,472,852, discussed infra.
Previously, we observed that at least a subpopulation of lupus anticoagulants can inhibit the APC anticoagulant activity more effectively than prothrombin activation (Smirnov, M. D., Triplett, D. T., Comp, P. C., Esmon, N. L., and Esmon, C. T. 1995. “On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies,” J. Clin. Invest. 94:309-316). This difference is dramatically augmented by the presence of PE in the membrane bilayer (Smirnov, M. D., Triplett, D. T., Comp, P. C., Esmon, N. L., and Esmon, C. T. 1995. J. Clin. Invest. 95:309-316; and Rauch, J., Tannenbaum, M., Tannenbaum, H., Ramelson, H., Cullis, P. R., Tilcock, C. P. S., Hope, M. J., and Janoff, A. S. 1986. “Human hybridoma lupus anticoagulants distinguish between lamellar and hexagonal phase lipid systems,” J. Biol. Chem. 261: 9672-9677).
In U.S. Pat. No. 5,472,852, entitled “Assay For Detection of Selective Protein C Inhibition by Patients” a method for determining the propensity of a patient to have a thrombotic incident was disclosed. A membrane source was utilized in the assay comprising an effective amount of phosphatidylethanolamine (PE) and an effective amount of phosphatidylserine (PS). Preferably, 10 to 50% PE and 5 to 50% of PS was utilized in the assay. Phosphatidylcholine (PC) could be used in the assay to make up any remaining percentage. In the assay disclosed and claimed in the '852 patent, patient and control plasma is assayed in the presence and absence of exogenous activated protein C (APC). By comparing the clotting times of samples with and without exogenous APC and optimal phospholipids, the risk of thrombotic disease can be assessed.
During the process of clot formation, leukocytes are recruited into the growing thrombus and become activated. These activated leukocytes have been reported to release potent oxidizing agents like hydrogen peroxide and superoxide, substances known to oxidize phospholipids. Malech, H. L. and Gallin, J. I. 1987. “Neutrophils in human diseases,” N. Engl. J. Med. 317:687-694; McCord, J. M. 1985. “Oxygen-derived free radicals in postischemic tissue injury,” N. Engl. J. Med. 312:159-163.
Although it has been established that phospholipids have an important role in coagulation, and coagulation assays such as discussed above, and some reports on specific parameters of the phospholipids have been reported as discussed above, there is a continuing need to improve assays for determining the risk of thrombotic disease by improving reagents used in such assays. In addition, although reports as to release of oxidizing agents from leukocytes have inferred that membrane oxidation may be involved in thrombosis, there has been no report or prediction of a differential effect of oxidation of lipids on APC activity.
It has now been found that oxidized phospholipids selectively enhance the anticoagulant properties of APC, with little impact on the clot-promoting reactions. It has also been found that a subset of antiphospholipid antibodies selectively eliminate the oxidized lipid enhancement. An assay is herein disclosed which is useful for assessing the risk of thrombotic episodes by utilizing oxidized and nonoxidized phospholipids as separate reagents. The sample is tested for clotting by using each of the reagents in a parallel assay. The results are compared to those obtained with normal plasma to assess whether the sample plasma may contain indicators of thrombotic disease.